Frequency facilitation, elicited by low-frequency stimulation (LFS) is a specific property of mossy fiber-CA3 synapses. Although it has been widely described in vitro, no evidence as yet exists as to whether this phenomenon occurs in vivo. Here, we show that, in freely behaving rats, frequency facilitation at mossy fiber-CA3 synapses consistently occurs in response to LFS (1 Hz). Extracellular adenosine regulates presynaptic neurotransmitter release via action on adenosine A1 receptors and contributes to frequency facilitation in vitro. We investigated whether adenosine A1 receptors mediate frequency facilitation in freely behaving animals. The adenosine A1 receptor antagonists, DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and phenylxanthine, markedly enhanced mossy fiber synaptic transmission and significantly occluded frequency facilitation. Evoked responses were suppressed by application of the group II metabotropic glutamate receptor agonist (2S,2ЈR,3ЈR)-2-(2Ј,3Ј-dicarboxycyclopropyl)glycine (DCG-IV), in line with the known sensitivity of mossy fiber-CA3 synapses to this agent. By comparison, neither frequency facilitation, effects of the adenosine A1 antagonists, nor effects of DCG-IV were evident at either perforant path-dentate gyrus synapses or commissural/associational CA3 synapses in vivo. These data support that frequency facilitation is an intrinsic property of information storage processes at mossy fiber-CA3 synapses in the intact animal and that frequency facilitation in vivo may be mediated by regulation of the adenosine A1 receptor.